The main focus of our work in this next year is to characterize by proton NMR the structures of trapped intermediates in the folding of ribonuclease A. The overall goal is to determine the kinetic pathway of folding in order to understand the mechanisms of folding. We have found previously that proline isomerization provides a kinetic barrier for trapping folding intermediates when slow-folding molecules containing a wrong cis or trans proline isomer refold at low temperatures (0 degrees-10 degrees C). In these conditions nearly complete folding occurs before the wrong proline isomer changes to the correct isomer, and intermediates also accumulate transiently at earlier stages of folding. We found last year that, when the exchangeable peptide N-H protons are (3H) labeled in the unfolded protein, a substantial number of the labeled protons are protected against exchange with solvent by the rapid formation of an early intermediate when refolding is initiated in conditions where exchange-out occurs rapidly from the completely unfolded protein. Probably H-bonded structure in the folding intermediate is responsible for protection against exchange. This hypothesis is being tested now by NMR studies of the protected protons, using H-D exchange to determine the extent of protection of individual protons. We expect to monitor stages in folding by finding the number of protected protons at each stage, and by measuring the extent of protection of each proton. In future work, assignment of these proton lines to known locations in the structure of RNaseA will provide a detailed description of the H-bonded structure at different stages in folding.